Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-08-13
2001-08-28
Martinell, James (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S325000, C435S252300, C536S023720
Reexamination Certificate
active
06281200
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of inflammatory responses and natural chemokine action. More particularly, it concerns the methods of making and using of novel chemokine-like genes and proteins from MCV type 1 and the closely related MCV type 2.
2. Description of Related Art
Poxviruses are notorious for their ability to evade the host's immune system by both active and passive mechanisms (Pickup, 1994). Since the eradication of smallpox, the only poxvirus that naturally infects humans is molluscum contagiosum virus (MCV). MCV causes benign proliferative lesions of the skin in normal and immunocompromised individuals. Persons with acquired immune deficiency syndrome (AIDS) sometimes get extensive MCV infections which can be disfiguring (Ray and Gateley III, 1994; Smith et al., 1994).
There are at least two types of MCV based on restriction endonuclease cleavage patterns of viral DNA (Darai et al., 1986; Porter et al., 1989; Thompson et al., 1990). Some epidemiological studies have been reported, but there is no consensus on anatomic or patient group distribution of the two MCV types. Although MCV cannot be propagated in tissue culture, the inventors and others have shown recently that MCV can be grown in human tissue implanted into immunodeficient mice (Buller et al., 1995; Fife et al., 1996).
Recently, much attention has been focused on chemokines, small proteins produced by lymphocytes and a variety of other cells. Chemokines were originally identified by their ability to attract inflammatory cells, but they have since been shown to have a variety of additional activities (for review, see Baggiolini et al., 1994). In addition to chemotaxis, some chemokines have been shown to cause decreased growth of hematopoietic stem and early subsets of myeloid progenitor cells (Broxmeyer et al., 1995; Broxmeyer et al., 1993; Broxmeyer et al., 1990; Graham et al., 1990) and some have been shown to block entry of human immunodeficiency virus into lymphocytes (Cocchie et al., 1995; Oberlin et al., 1996). Roles for chemokines in a variety of allergic and autoimmune diseases have also been postulated, although direct evidence is limited (for review see Mantovani et al., 1996).
It has been assumed that MCV, like other poxviruses, has immune evasion functions. The recent report of the nucleotide sequence of the MCV genome (Senkevich et al., 1996) has permitted identification of potential candidate viral proteins that may be involved in escape from the host immune system. One such putative viral protein is designated MC148R. This open reading frame potentially encodes a 104 amino acid protein with significant homology to &bgr; chemokines such as macrophage inflammatory protein (MIP)-1&bgr; (SEQ ID NO:6). The putative MC148R protein contains a five amino acid deletion at the functionally important amino terminus of the mature protein, relative to MIP-1&bgr; (Senkevich et al., 1996). Only three other viruses, human herpes virus 6 (Gompels et al., 1995), human herpes virus 8 (Moore et al., 1996; Nicholas, 1997) and murine cytomegalovirus (MacDonald et al., 1997) are known to encode chemokine analogs. These herpesvirus chemokine analogs do not contain deletions, although there is only limited information about their ability to function as chemokine agonists.
There are important issues regarding this MCV viral protein that need to be resolved. It needs to be determined whether this protein may act as a chemokine agonist, if so, are there other chemokines agonists expressed by MCV. The pathogenic role of MCV viral proteins play is unknown. Further there is little information on how MCV infection escapes immune attack from the host, the presence of MCV viral protein and other proteins related to this protein may somehow effect immune system escape. The answers to these question would have many benefits in the treatment of viral infection as well as effecting immune escape in cells in which it would be desirable to suppress the host immune system.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified chemokine-like protein. In a particular embodiment the preferred polypeptide has the sequence of SEQ ID NO:2. In yet another embodiments the preferred polynucleotide has the sequence of SEQ ID NO: 1. In certain embodiments, the present invention provides a peptide of about 10 to about 50 contiguous amino acids of SEQ ID NO:2. In preferred embodiments, the peptide is about 10 amino acids in length. In other preferred embodiments, the peptide may be independently, about 15 amino acids in length, about 20 amino acids in length, about 25 amino acids in length, about 50 amino acids in length or about 100 amino acids in length.
The present invention also provides an isolated and purified nucleic acid encoding the chemokine-like protein having an amino acid sequence as set forth in SEQ ID NO:2. In particularly preferred embodiments, the nucleic acid sequence is as set forth in SEQ ID NO: 1 or the complement thereof. In certain embodiments, the present invention provides an oligonucleotide of about 15 to about 50 contiguous base pairs of SEQ ID NO: 1, or the complement thereof. In other preferred the oligonucleotide may be independently about 15 base pairs in length, about 20 base pairs in length, about 25 base pairs in length, about 50 base pairs in length, about 100 base pairs in length or about 200 base pairs in length
A purified antibody that is immunoreactive with a chemokine-like MCV viral protein is also provided by the present invention. In particular embodiments, the MCV viral protein has an amino acid sequence as set forth in SEQ ID NO:2, in other embodiments, the MCV viral protein has an amino acid sequence as set forth in SEQ ID NO:4.
In another aspect of the present invention there is provided an expression construct comprising a vector comprising an isolated polynucleotide encoding a chemokine-like MCV viral protein and a promoter operably linked to the isolated polynucleotide.
In preferred embodiments, the vector may be a viral vector. In preferred embodiments, the viral vector may be selected from the group consisting of a retroviral vector, an adenoviral vector, a herpesviral vector, adeno-associated viral vector and a cytomegaloviral vector. In other embodiments, the viral vector further comprises a polyadenylation signal. In other preferred embodiments, the expression construct encodes an MCV viral protein that has an amino acid sequence as set forth in SEQ ID NO:2. In other embodiments, the MCV viral protein has an amino acid sequence as set forth in SEQ ID NO:4.
In specific embodiments, the promoter is selected from the group consisting of CMV IE, SV40 MLP, AdE1, keratin 10 promoter, baculovirus polyhedrin promoter and &bgr;-actin.
Also provided is a recombinant host cell comprising with a vector comprising an expression region encoding a chemokine-like MCV viral protein operatively linked to a promoter.
The present invention also describes a method for treating MCV induced proliferative lesions of the skin in an individual comprising inhibiting the activity of chemokine-like MCV viral protein in the lesion. In particular aspects, the inhibiting comprises contacting the lesion with an inhibitor of chemokine-like MCV viral proteins. In certain embodiments, the inhibitor comprises a vector comprising an expression cassette comprising a gene encoding MCV viral protein placed antisense to and operatively linked to a promoter; wherein expression construct inhibits the function of chemokine-like MCV viral protein. In other embodiments, the method may further comprise administering to the individual an inhibitor of chemokine-like MCV viral protein activity identified according to a method comprising the steps of (i) providing a cell expressing an MCV viral protein; (ii) contacting the cell with a candidate substance; (iii) measuring the activity of the MCV viral protein in the cell; and (iv) comparing the activity of the protein in the cell of step (iii) with the activity of the protein in the c
Brown Darron R.
Broxmeyer Hal E.
Fife Kenneth H.
Hromas Robert
Krathwohl Michell D.
Advanced Research & Technology Institute
Fulbright & Jaworski
Martinell James
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